View : 532 Download: 0

Full metadata record

DC Field Value Language
dc.contributor.author노은진-
dc.creator노은진-
dc.date.accessioned2016-08-25T04:08:11Z-
dc.date.available2016-08-25T04:08:11Z-
dc.date.issued2005-
dc.identifier.otherOAK-000000011579-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/178977-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000011579-
dc.description.abstract아밀로이드 전구단백질(amyloid precursor protein: APP)은 내재성 막 당 단백질(integral membrane glycoprotein)로서, 대사과정 중 아밀로이드 펩티드 (amyloid-α peptide: Aβ)를 생성한다. 아밀로이드 펩티드는 기억력 저하, 인지능력 저하, 주의력 저하, 사고력 저하와 판단력 저하 등의 임상적 증상과 대뇌피질과 해마 또는 편도핵에서 neurofibrillary tangle과 senile plaque이 관찰되는 병리학적 증상을 나타내는 신경퇴행성 질환의 일종인 Alzheimer disease (AD)에 있어서 학습과 기억 능력에 손상을 주는 요인으로 작용하는 것으로 알려져 있다. 이에 반해, 정상적인 세포에서 APP의 대사 과정 중에 생성되는 용해성 아밀로이드 전구단백질 (soluble amyloid precursor protein: sAPPα)는 세포 밖으로 분비되어 세포의 생존, 증식과 부착을 조절하며 신경돌기의 발아 후 생육을 유도한다. APP의 분해과정에 대한 많은 연구가 행하여져 왔고, 그 과정에 무스카린성 아세틸콜린 수용체가 관계한다는 사실이 보고되었으나, 무스카린성 아세틸콜린에 의한 sAPPα의 정확한 유리조절기전에 대해서는 아직 자세히 밝혀져 있지 않다. 본 연구에서는 이러한 무스카린성 수용체 활성화에 의한 sAPPα 유리 증가의 신호 전달 기전을 밝히고자 했다. 특히, M1, M3 수용체가 모두 존재하는 SH-SY5Y 셀라인에서 PI3K/Akt, NFkB, TRPC1의 역할을 연구하였다. 최근 우리 연구에서 세포 내 Ca2+ 저장소의 고갈을 보충하기 위한 capcitative calcium entry (CCE)가 무스카린성 수용체 활성에 의해 유도되는 sAPPα의 유리 증가에 관여한다는 것을 밝힌 바 있다. 본 연구에서는, SH-SY5Y 셀라인에서 무스카린성 수용체 활성에 의해 유도되는 sAPPα의 유리와 CCE의 조절에 PI3-kinase/Akt의 역할을 탐구해보았다. 전의 연구에서, PI3-kinase inhibitor인 wortmannin과 LY294002를 처리했을 때, Ca2+ influx와 sAPPα의 유리는 크게 감소된 것을 관찰하였다. 본 연구에서, dominant negative PI3-kinase construct를 transfection하면 sAPPα의 유리가 감소하는 것을 관찰함으로써 PI3-kinase의 역할을 뒷받침했다. PI3-kinase를 통한 세포생존에 관여하는 것으로 알려져 있는 Akt가 sAPPα와 CCE에 미치는 영향을 확인하기 위해, wild type (wt) Akt와 dominant negative인 Akt K179M 및 PI3-kinase와 interaction하지 않는 mutant인 Akt R25C가 SH-SY5Y에 transfection 되었다. wtAkt가 transfection된 셀에서는 무스카린성 수용체 활성에 따른 Akt kinase의 활성, sAPPα과 CCE가 크게 증가한 반면, Akt K179M과 Akt R25C가 transfection된 셀에서는 감소하여, Akt가 무스카린성 수용체 활성에 의한 sAPPα의 유리와 CCE에 관여하고 있음을 알 수 있다. 우리는 이전 연구에서 무스카린성 수용체 활성에 의한 sAPPα의 유리에 관여하는 CCE channel로 알려져 있는 transient receptor potential canonical (TRPCs)은 SY-SY5Y 셀에서 subtype 1만 존재하는 것으로 밝힌 바 있다. 따라서 본 실험은 TRPC1을 transfection함으로 진행되었는데, 무스카린성 수용체 활성을 통한 CCE는 transfection된 TRPC1의 양에 비례해 증가했으며, 이에 따른 sAPPα 유리 또한 증가했다. 이러한 결과들은 SH-SY5Y 셀에서 TRPC1을 통한 extracellular Ca2+ influx가 무스카린성 수용체 활성에 따른 sAPPα을 조절할 수 있음을 의미한다. CCE와 NF-κB와 관련된 우리의 이전 실험에서 무스카린성 수용체 활성에 따른 CCE는 NF-κB 활성의 upstream이라는 것을 밝혔다. 본 실험에서 무스카린성 수용체 활성에 따른 TRPC1과 NF-κB의 관계를 밝히기 위해, TRPC1이 과발현된 SH-SY5Y 셀에서 NF-κB inhibitor인 TMB-8, [6-amino-4-(4-phenoxyphenylethylamino)quinazoline, QNZ], MG132, 및 BAY11-7085가 oxoM을 통한 sAPPα 유리에 어떤 영향을 미치는지 관찰하였다. TRPC1이 과발현된 셀에서 NF-κB는 증가하였고, 예상되다시피 이러한 NF-κB inhibitor들은 sAPPα의 유리를 크게 감소시켰다. 이 결과로부터 TRPC1이 무스카린성 수용체 활성에 의한 sAPPα의 유리에 관련된 NF-κB 활성 경로의 upstream이라는 것을 알 수 있다. 무스카린성 수용체 활성에 따른 sAPPα의 유리조절기전을 설명하는 이 연구는 AD의 병리학에 관여하는 콜린성 체계을 이해하는데 도움을 줄 것이다.;The amyloid precursor protein (APP) is a transmembrane protein that undergoes proteolytic cleavage to produce Aβ sequence, found in the brains of Alzheimer's disease (AD). If processed by an unidentified enzyme designated α-secretase it liberates a soluble N-terminal fragment (sAPPα). Biological activities of sAPPα have been shown to include promotion of neuronal cell survival, adhesive interactions, neurite outgrowth, synaptogenesis, and synaptic plasticity. Neurotransmitters, hormones, or cytokines, as well as other neuroactive compounds that activate PKC and other transduction signals, increase secretion of sAPPα via the nonamyloidogenic pathway. The release of soluble amyloid precursor protein (sAPPα) produced from α-secretase processing by cleavage within the amyloid β-peptide domain of APP is highly regulated by several external and internal signals. The Gq protein-coupled muscarinic (M1 & M3) receptors are known to regulate sAPPα release. However, their signaling mechanism is not clearly understood. The purpose of this thesis was to uncover the signaling mechanism for the regulation of sAPPα release mediated by muscarinic receptor activation. Especially the involvement of PI3K/Akt, NF-κB, and transient receptor potential (TRP) pathway are examined in human neuronal cell line, SH-SY5Y, which endogenously expresses both M3 and M1 muscarinic receptor subtypes. Recently we have shown that capacitative Ca2+ entry (CCE), a refilling mechanism for depleted intracellular calcium stores, is involved in the regulation of muscarinic receptor mediated sAPPα release. In the present study, the role of PI3-kinase/Akt signaling pathways in regulating muscarinic receptor-mediated sAPPα release and CCE was investigated in SH-SY5Y cells. In our previous study, when cells were treated with PI3-kinase inhibitors, wortmannin and LY294002, muscarinic agonist-stimulated Ca2+ influx and sAPPα release was significantly inhibited. In the present study, the role of PI3-kinase was further confirmed by transfection of a dominant negative form of PI3-kinase, which reduced sAPPα release. To define the role of Akt, known as a regulator of PI3-kinase-mediated cell survival, in regulating muscarinic receptor-mediated sAPPα release and CCE, wild type (wt) Akt, dominant negative Akt K179M mutant and AktR25C mutant lacking interaction with PI3-kinase, were transfected into SH-SY5Y cells. In wtAkt transfected cells, muscarinic receptor stimulation markedly increased Akt kinase activities, sAPPα release, and CCE, whereas in cells transfected with AktK179M or AktR25C, significant inhibitory effects were observed, indicating that Akt may be involved in muscarinic receptor-mediated sAPPα release pathway and CCE. In our previous study, we tried to identify the CCE channel responsible for the regulation of muscarinic receptor mediated sAPPα release. The transient receptor potential canonical (TRPCs) have been understood as channel candidates for the CCE channels. We previously examined that only TRPC 1 mRNA is expressed in SH-SY5Y cells among TRPC 1-7. Experiments were processed in cells transfected transiently with TRPC1 in which TRPC1 gene was overexpressed in a concentration-dependent manner. We observed that the CCE induced by muscarinc receptor agonist was increased in cells transfected transiently with TRPC1. sAPPα release evoked by muscarinic receptor agonist was also significantly increased in cells transfected transiently with TRPC1. These results suggested the possibility that muscarinic receptor-mediated extracellular Ca2+ influx via TRPC1 can modulate muscainic receptor-mediated sAPPα release in SH-SY5Y cells. Our previous results concerning NF-κB and CCE indicated that the CCE induced by muscarinic receptor activation is upstream of NF-κB activation pathway, which is involved in muscarinic receptor-mediated sAPPα release. To investigate the relationship between TRPC1 and NF-κB by stimulation of muscarinic receptor, we examined how sAPPα induced by oxoM responds to various NF-κB inhibitors SY-SY5Y cells transfected transiently with TRPC1. A selective NF-κB activation inhibitor, [6-amino-4-(4-phenoxyphenylethylamino)quinazoline, QNZ], TMB-8, MG132, and BAY11-7085 were used. NF-κB was increased in cells transfected transiently with TRPC1 and, as expected, these NF-κB inhibitors significantly decreased sAPPα release. These results indicate that TRPC1 is upstream of NF-κB activation pathway which is involved in muscarinic receptor-mediated sAPPα release. This study on the mechanisms explaining the muscarinic receptor-mediated sAPPα release may lead to a better understanding of the significance of the cholinergic system in the pathophysiology of AD.-
dc.description.tableofcontentsAbstract = i 1. Introduction = 1 2. Materials & Methods = 18 2.1. Materials = 18 2.2. Mammalian cell culture = 19 2.3. Measurement of sAPPα release = 19 2.3.1. Stimulation and inhibition of sAPPα release = 19 2.3.2. Measurement of sAPPα release by SDS-PAGE and Western blotting = 20 2.4. Measurement of changes in [Ca2+]i = 21 2.5. Identification of TRPC1 mRNA reverse transcriptase polymerase chain reaction = 22 2.6. Transfection of SH-SY5Y cells by electroporation = 23 2.7. Akt activity assay = 25 3. Results = 28 3.1. Involvement of PI3-kinase in muscarinic receptor-mediated sAPPα release = 28 3.2. Regulation of muscarinic receptor-mediated sAPPα release by Akt = 32 3.2.1. Kinase activity of Akt mustants = 35 3.2.2. Akt activation by muscarinic receptor = 35 3.2.3. Effects of Akt mutants on muscarinic receptor-mediated extracellular Ca2+ influx and sAPPα release = 37 3.3. Regulation of muscarinic receptor-mediated sAPPα release by transient receptor potential canonical 1 (TRPC1) = 40 3.3.1. Overexpression of TRPC1 gene = 40 3.3.2. Muscarinic receptor-mediated extracellular Ca2+ influx and sAPPα release in TRPC1-overexpressed SH-SY5Y cells = 44 3.4. Involvement of NF-κB activation in regulation of muscarinic receptor-mediated sAPPα release in TRPC1 overexpressed cells = 47 4. Discussion = 51 5. Referenes = 59 국문초록 = 76-
dc.formatapplication/pdf-
dc.format.extent1451924 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.titleInvolvement of PI3K/Akt, NF-kB and TRPC1 in Regulation of Muscarinic Receptor-mediated sAPPα Release-
dc.typeMaster's Thesis-
dc.format.pageviii, 78-
dc.identifier.thesisdegreeMaster-
dc.identifier.major대학원 약학과-
dc.date.awarded2006. 2-
Appears in Collections:
일반대학원 > 생명·약학부 > Theses_Master
Files in This Item:
There are no files associated with this item.
Export
RIS (EndNote)
XLS (Excel)
XML


qrcode

BROWSE